Hybrid 3′ untranslated regions suitable for efficient protein expression in mammalian cells

ABSTRACT

The present invention describes the use of hybrid short 3′ untranslated (3′UTR) regions which are composed of two regions, one region from an 3′ untranslated region of a stable eukaryotic mRNA, and another region from the downstream end of an 3′ untranslated region of another eukaryotic mRNA that contains a polyadenylation (polyA) signal. The use of such hybrid regions allows for an efficient protein expression when used in conjunction with circular or linear expression DNA molecules. The present invention provides a recombinant DNA that is composed of a promoter, a protein coding region, and the hybrid 3′UTR in a continuous and directional orientation. The efficient expression systems of the invention are suitable for the economical and efficient production of therapeutic proteins and for use with both transient and stable expression systems.

FIELD OF INVENTION

The field of invention is expression vector engineering, mammalian protein expression, recombinant DNA technology, and expression of therapeutic proteins.

BACKGROUND OF INVENTION

Eukaryotic messenger RNA (mRNA) contains three regions, 5′ untranslated region (5′UTR), protein coding region, and 3′ untranslated region (3′UTR). The 3′UTR is the sequence towards the 3′ end of the mRNA and contains a polyadenylation signal and a polyadenylation site. The 3′UTR is an important regulatory element in many instances it dictates the mRNA stability and it can also regulate translation efficiency. For example, AU-rich elements tend to destabilize mRNAs (Dalphin et al. 1999). The mRNAs can be classified into three categories based on their half-life: (a) unstable mRNAs with a short half life, for example, less than 2 hours, and (b) intermediately stable mRNAs with a half life that exceeds, for example, 2 hours, and (c) stable mRNAs with a half life that exceeds 8 hours. Many of the housekeeping genes tend to code for stable mRNAs, such as but not limited to globin mRNAs (Chen and Shyu 1994; Shaw and Kamen 1986), β-actin, ribosomal proteins, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

Expression vectors are important research and biotechnology tools. In research they are used to study protein function while in industry they are used to produce (therapeutic) proteins. Optimization of expression vectors for efficient protein production has been sought and practiced in many ways, mostly by optimizing strong promoters that lead to an efficient transcription of the mRNA encoded by the protein coding region in the expression vector. Examples of these are the CMV immediate early promoter, SV40 promoter, elongation factor (EF) promoter, and chicken β-actin promoter (Foecking and Hofstetter 1986; Kobayashi et al. 1997).

Likewise, strong polyA signal cassettes were also used to augment the protein expression by providing strong polyadenylation signals that stabilize mRNAs which are produced from the expression vectors. Among the widely used polyA signals are the bovine growth hormone (bGH) polyA signal (U.S. Pat. No. 5,122,458 by Post et al.: Use of a bGH GNA polyadenylation signal in expression of non-bGH polypeptides in higher eukaryotic cells) and SV40 polyA signal (Goodwin and Rottman 1992). Unlike termination signals in bacteria, where the 3′ end of the mRNA is formed by the NA polymerase that is simply dropping off the DNA and ceasing transcription, in eukaryotes cleavage occurs that is followed by binding of the polyadenylation complex to an AAUAAA sequence near the end of the mRNA. This complex contains an endonuclease that cuts the RNA about 14-30 bases downstream of the AAUAAA sequence and a polymerase that adds a string of polyA forming the polyA tail (Wigley et al. 1990).

Since the 3′ UTR and polyA signal context sequence may influence nuclease degradation of plasmid DNA vectors particularly after delivery and during trafficking to the nucleus, a novel approach that circumvents this problem is described. In this invention, an approach to further optimize expression of proteins is described which uses a hybrid 3′UTR downstream of the protein coding region that consists of two regions, wherein one region is from an 3′UTR of a stable mRNA and the other region is a poly A signal containing region from an 3′UTR of another mRNA.

SUMMARY OF INVENTION

The present invention describes the construction of hybrid short 3′ untranslated (3′UTR) regions for use in mammalian expression vectors to boost the expression of proteins encoded in these vectors. The hybrid short 3′UTR comprises two regions that are near each other, i.e. in proximity to each other or adjacent to each other or overlapping with each other or one encompasses the other, one region from an 3′ untranslated region of a stable eukaryotic NA, and another region from the downstream end of an 3′ untranslated region of a stable eukaryotic mRNA which contains the polyadenylation signal. The first region does not contain a polyadenylation signal, whereas the second region contains a polyadenylation signal and is derived from the downstream end of an 3′ untranslated region of a stable eukaryotic mRNA.

In a preferred embodiment, the first region or the two regions comprise the 3′UTR or part of the 3′UTR of the human eukaryotic translation elongation factor 1 alpha 1 EEF1A1).

The present invention further provides a recombinant DNA that is composed of promoter, protein coding region, and the hybrid 3′UTR, in a continuous and directional orientation.

BRIEF DESCRIPTION OF FIGURES

FIG. 1. Construction of the hybrid 3′UTR construct. A vector containing CMV/Itron A promoter, a coding region for green fluorescence protein (GFP), followed by an 3′UTR containing an eukaryotic polyadenylation signal (AATAAA), is modified to include part of the 3′UTR of the human housekeeping gene, human eukaryotic translation elongation factor 1 alpha 1 (EEF1A1). The EEF1A region was cloned into the original 3′UTR region using restriction site cloning and the result is a hybrid 3′UTR region. The upper panel A shows the original construct and the lower panel B shows the new construct with the hybrid 3′UTR.

FIG. 2. GFP fluorescence due to protein expression in HEK 293 cells using the unmodified vector (A) and vector containing the hybrid 3′UTR (B).

FIG. 3. GFP fluorescence due to protein expression in two different cell types, HeLa (A and B) and Huh7 liver cell line (C and D) using the unmodified vector (A and C) and vector containing the hybrid 3′UTR (B and D).

FIG. 4. GFP fluorescence due to protein expression in HEK 293 cells using the unmodified vector (A) and linear DNA containing the hybrid 3′UTR in form of a PCR product (B).

DETAILED DESCRIPTION OF INVENTION

As discussed above, there is a need to improve and optimize protein expression. Therefore, the problem to be solved by this invention, was to further improve and optimize protein expression systems, such as by improving and optimizing the engineering of expression vectors and/or of their components.

The problem is solved by the present invention by providing a recombinant nucleic acid that comprises a first and a second nucleic acid region, wherein

-   -   (a) said first region is derived from an 3′ untranslated region         (3′UTR) of a stable eukaryotic mRNA,     -   (b) said first region does not contain a polyadenylation signal,     -   (c) said second region is derived from the downstream end of an         3, untranslated region of another or the same stable eukaryotic         mRNA as in (a), and     -   (d) said second region contains a polyadenaylation signal.

With the proviso that said first and second nucleic acid regions when taken together form a hybrid 3′UTR, said hybrid 3′UTR being different from its naturally occurring, i.e. original, counterpart. This proviso means, for example, when said first region is derived from the upstream end of a 3′-UTR of a stable eukaryotic mRNA and said second region is derived from the downstream end of the 3′UM of the same stable eukaryotic mRNA, the hybrid 3′UTR is different from the naturally occurring, i.e. original, 3′UTR.

By “nucleic acid” is meant a single-stranded or double-stranded chain of two or more nucleotide bases including, without limitation, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and analogs of either DNA or RNA, mRNA, and cDNA, such as PNA. The recombinant nucleic acid of the present inventions are preferably DNA, RNA, or PNA.

By the term a region is “derived from” an 3′ untranslated region (3′-UTR) of a stable eukaryotic mRNA is meant that the region is the 3′UTR itself is a part of said 3′UTR or a sequence of the 3′UTR comprising mutations, deletions, truncations, insertions and/or single nucleotide polymorphisms (SNPs).

In a preferred embodiment recombinant nucleic acids are provided, wherein said first region and said second region of the recombinant nucleic acid are in proximity to each other or wherein said first region and said second region are adjacent to each other or wherein said first region and said second region overlap with each other or wherein one region encompasses the other region.

Preferred proximities of the first and second region, i.e. distances between the first and second region, are in the range of 0 to 500 nucleotides, more preferred in the range of 0 to 250 nucleotides and most preferred in the range of 0 to 100 nucleotides.

It is preferred that the 3, end of the first region is adjacent to the 5′ end of the second region or that the 3′ end of the second region is adjacent to the 5′ end of the first region.

It is further preferred that the first region is located with the second region, wherein the first region is preferably located upstream of the polyadenylation signal of the second region.

It is preferred that at least one of the regions is derived from stable mRNAs, such as those of housekeeping genes which exist in abundant amounts inside the cells. As already defined above, stable mRNAs are mRNAs with a half life that exceeds 8 hours.

Preferred housekeeping and/or stable mRNAs are, but are not limited to β-actin, α- and β-globins, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), growth hormone, eukaryotic translation elongation factor 1 alpha 1 (EEF1A1), and many of the ribosomal proteins. Other housekeeping genes are known in the art, see for example Eisenberg and Levanon 2003. It is preformed that the first region and/or the second region is derived from the 3′UTR of housekeeping genes that are abundant (see Table 1).

Since the length of the 3′UTR of housekeeping genes is generally shorter, see Eisenberg and Levanon, 2003, which is also the result of our analysis, see Example 1, the preferred length of the total hybrid 3′UTR according to the invention is less than 1000 nucleotides, preferably less than 500 nucleotides.

In a preferred embodiment the first region is derived from the 3′UTR of eukaryotic translation elongation factor 1 alpha 1 (EEF1A1), see also FIG. 1 and Table 2. In a more preferred embodiment the first region is the 3′UM of eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) or a part thereof.

In another preferred embodiment the first and the second region is derived from the 3′UTR of eukaryotic translation elongation factor 1 alpha 1 (EEF1A1).

In another preferred embodiment, the first region is derived from the 3′UTR of EEF1A1 and the second region is derived from 3′UTR of bovine growth hormone (bGH), preferably a smaller region of the bovine growth hormone 3′UTR that ter contains an efficient polyadenylation signal.

In another aspect of the present invention a recombinant nucleic acid is provided that comprises a first and a second nucleic acid region, wherein both regions are derived from the 3′UTR of the same stable eukaryotic mRNA, preferably from the 3′UTR of eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) or a part thereof. Furthermore, in this aspect of the present invention the above stated proviso does not apply. In a preferred embodiment of this aspect of the present invention the first or second region is identical to the 3′UTR of EEF1A1 or one or more parts thereof. In another preferred embodiment of this aspect of the present invention the first and second region when taken together are identical to the 3′-UTR of EEF1A1 or one or more parts thereof.

The problem is further solved by the present invention by providing a further recombinant nucleic acid comprising the following components in 5, to 3′ direction:

-   -   (i) a promoter sequence,     -   (ii) a protein coding region sequence, and     -   (iii) the hybrid 3′UTR of the present invention as defined         above, which is operably lined to the protein coding sequence of         (ii).

In another aspect of the present invention the further recombinant nucleic acid comprises the following components in 5′ to 3′ direction:

-   -   (i) a promoter sequence,     -   (ii) a protein coding region sequence, and     -   (iii) the 3′UTR of EEF1A1 or parts thereof as defined above,         which is operably linked to the protein coding sequence of (ii).

By “operably linked” is meant that the nucleic acid sequence encoding a protein of interest and (transcriptional) regulatory sequences, such as the 3′UTR, are connected in such a way as to permit expression of the nucleic acid sequence in vivo, such as when introduced into a cell. Preferably, the hybrid 3′UTR of the present invention is operably lined to the protein coding sequence in a continuous (uninterrupted) and directional orientation, such as adjacent to the protein coding sequence.

In a preferred embodiment the recombinant nucleic acid of the invention is a linear DNA molecule. Such linear DNA molecules are preferably generated by PCR using at least two primers that specifically hybridize to regions near the 5′ end and specifically hybridize to regions near the 37 end of said nucleic acid which is comprised in an expression vector.

The problem is further solved by the present invention by providing an expression vector comprising the nucleic acid according to the present invention.

It is preferred that the expression vector further comprises a selection marker. Selection markers are known in the art however, preferred selection markers are neomycin resistance gene and blasticidin resistance gene.

The protein coding sequences are either coding for a reporter gene or more preferably a therapeutic protein. Preferred reporter genes are GFP, luciferase, or other reporter genes known in the art. Preferred therapeutic proteins are interferones, growth factors, anti-angiogenesis proteins, apoptosis modulating proteins, tumor growth factors or spread suppressing factors, vaccines, recombinant antibodies, or any other current or future therapeutic protein.

The nucleic acids according to the present invention are preferably produced in linear form by a method that comprises the following steps of

-   -   (a) providing an expression vector comprising a nucleic acid         according to the present invention, wherein said nucleic acid         comprises the following components in 57 to 3′ direction:         -   (i) a promoter sequence,         -   (ii) a protein coding region sequence, and         -   (iii) the hybrid 3′UTR of the present invention as defined             above, which is operably linked to the protein coding             sequence of (ii),     -   (b) providing at least two primers, wherein one primer         specifically hybridizes to regions near the 5, end of the         nucleic acid according to the present invention, and         wherein the other primer specifically hybridizes to regions near         the 3′ end of the nucleic acid according to the present         invention,         wherein said nucleic acid comprises the following components in         5, to 3′ direction:     -   (i) a promoter sequence,     -   (ii) a protein coding region sequence, and     -   (iii) the hybrid 3′UTR of the present invention as defined         above, which is operably linked to the protein coding sequence         of (ii),     -   (c) performing a PCR, and     -   (d) obtaining the amplification product of step (c).

In another aspect of the invention the nucleic acids according to the present invention are preferably produced in linear form by a method that comprises the following steps of

-   -   (a) providing an expression vector comprising a nucleic acid         according to the present invention, wherein said nucleic acid         comprises the following components in 5′ to 3, direction:         -   (i) a promoter sequence,         -   (ii) a protein coding region sequence, and         -   (iii) the 3′UTR of EEF1A1 or parts thereof as defined above,             which is operably linked to the protein coding sequence of             (ii),     -   (b) providing at least two primers, wherein one primer         specifically hybridizes to regions near the 5′ end of the         nucleic acid according to the present invention, and wherein the         other primer specifically hybridizes to regions near the 3′ end         of the nucleic acid according to the present invention,     -   wherein said nucleic acid comprises the following components in         5′ to 37 direction:         -   (i) a promoter sequence,         -   (ii) a protein coding region sequence, and         -   (iii) the 3′UTR of EEF1A1 or parts thereof as defined above,             which is operably linked to the protein coding sequence of             (ii)     -   (c) performing a PCR, and     -   (d) obtaining the amplification product of step (c).

The problem is further solved by the present invention by providing a host cell characterized in that it contains the expression vector of the present invention or the recombinant linear nucleic acid of the present invention, and further characterized in that it transiently or stably expresses the protein encoded in the expression vector of the present invention or the recombinant linear nucleic acid of the present invention.

Transient and stable expression of proteins is known in the art. When proteins are expressed transiently a foreign gene that codes for the particular protein is expressed by recipient/host cells over a relatively brief time span, wherein the gene is not integrated into the genome of the host cell, whereas in case of stable expression of proteins the foreign coding gene is integrated into the genome of the host cell.

The host cells of the present invention are preferably obtained by in vivo injection of the expression vector of the present invention or the recombinant linear nucleic acid of the present invention into cells.

A preferred method for obtaining a host cell of the present invention comprises the following steps of

-   -   (a) providing an expression vector of the present invention or a         recombinant linear nucleic acid of the present invention,     -   (b) in vivo injection of the expression vector of the present         invention or the recombinant linear nucleic acid of the present         invention into cells,     -   (c) obtaining the injected cells of step (b).

Suitable cells are known in the art. However, preferred cells are HEK 293, HeLa, Huh7, COS-7, and CHO cell lines.

A preferred embodiment is a method for expressing proteins, comprising providing and culturing of host cells of the present invention, under conditions allowing transient or stable expression of proteins, and obtaining said expressed proteins.

Culturing conditions of cells and cell culturing conditions that allow the expression of proteins are known in the art. Preferred conditions are at temperature of about 37° C. and a humidified atmosphere, a medium containing buffers with a salt concentration and a pH1 suitable for mammalian cell culture.

The proteins can be either expressed in a secreted form or in cellular compartments. The expressed proteins can further be obtained by methods for isolating and purifying proteins from cells, which are known in the art. Preferred methods for purification are affinity chromatography, immuno-affinity chromatography, protein precipitation, buffer exchanges, ion-exchange chromatography, hydrophobic interaction chromatography, size-exclusion chromatography and electrophoresis-based purification.

Mammalian expression vectors are powerful research and industrial biotechnology tools. They comprise as a minimum requirement an eukaryotic promoter, a protein coding sequence, and an 3′UTR that contains an efficient polyadenylation signal. Many attempts of enchancing protein expression from vectors have been focused on promoters. In this invention, enhanced efficiency of protein expression was achieved by modifying the 3′-UTR that is continuously and directionally operable linked to the protein coding sequence.

The 3′UTRs which include the polyadenylation signal and upstream and downstream polyadenylation context sequences can be rendered efficient by making a hybrid 3′UTR consisting of two regions: a first region from an 3′ untranslated region of a stable eukaryotic mRNA that does not contain a polyadenylation signal, and a second region from the downstream end of an 3′ untranslated region of a stable eukaryotic mRNA that contains a polyadenylation signal.

The sequences can be obtained by amplification of the said region from the 3′UTR of the housekeeping mRNA using RT-PCR utilizing two primers specific to the region of interest. These PCR products are ten cloned into a vector, such as a plasmid or viral vector, which contains already the second region. Preferred examples for vectors are pUC 19 based plasmids, pcDNA3.1, Gwiz, CMVSport, etc. which are available from different research tool companies, such as invitrogen, Clontech, Strategene and Gene Therapy Systems, inc. There are different preferred ways of cloning, such as restriction site-directed cloning, blunt cloning, or recombination-based cloning. The hybrid USR can preferably be created by conventional cloning techniques involving restriction enzyme digestion of commercially available plasmids and cDNA molecules, or can be synthesized using PCR or an automated DNA synthesizer using methods known in the art.

It has been shown that a large fraction of human polyadenylation sites is flanked by U-rich elements, both upstream and downstream of the cleavage site, located around positions 0 to −50 and +20 to +60, relative to the polyA signal (Legendre and Gautheret 2003) Thus, it is likely that the enhanced efficiency of protein expression which is achieved by the present invention is caused by the presence of additional strong upstream sequences in the 3′UTR of EEF1A1.

Once the expression vectors containing the hybrid molecules are generated they can be transfected into cells by any known transfection method. Alternatively, linear fragments can be generated by PCR or synthesized genes that contain the minimum linear cassette containing the promoter, coding region, and the hybrid 3′UTR.

EXAMPLES

The following examples are intended for illustration purposes only, and should not be construed as limiting the scope of the present invention in any way.

Example 1

We used the list of housekeeping genes that were identified by Eisenberg and Levanon (2003) based on constitutive expression in all tissues. We assumed that a housekeeping gene with an abundance of more than 1000 ESTs (total number of expressed sequence tags in EST database per expressed genes) is stable because abundant transcripts are likely to be associated with stable mRNAs. The abundance data were obtained from the dbEST, a database of expressed sequence tags that is publicly available in the National Center for Biotechnology Information (NCBI, USA). Table 1 below shows the list of these abundant housekeeping genes and this list also constitutes the preferred sequence data source for the hybrid 3′UTR used in this invention. When compared to our list of unstable mRNA (derived from AU-rich mRNA which tend to code for unstable mRNAs), we found different statistical differences. The average abundance of housekeeping mRNAs was 1151±78 (SEAM n=570), i.e. higher than those of unstable mRNAs (by 5-fold) which had an average abundance of 220±16 (n=266). The average length of the 3′UTR of stable mRNAs appears shorter than those of unstable mRNAs (AU-rich mRNAs). The average length of 3′UTR of the housekeeping mRNA was 676±30 nucleotides (SEM, n±572) while the average length of the 3′UTR of abundant housekeeping mRNA, i.e., those with more than 1000 ESTs, was 570±50 nucleotides (SEM, n=178). In contrast, the average length of unstable AU-rich mRNAs was more than 1560±39 nucleotides (SEM, n=1027).

TABLE 1 List of Abundant Housekeeping Genes Acc Definition Symbol^(a) Length^(b) Abundance^(c) NM_001402 Eukaryotic translation elongation factor 1 alpha 1 EEF1A1 387 20011 NM_001614 Actin, gamma 1 ACTG1 718 16084 NM_002046 Glyceraldehyde-3-phosphate dehydrogenase GAPD 201 15931 NM_001101 Actin, beta ACTB 593 15733 NM_000967 Ribosomal protein L3 RPL3 74 10924 NM_006082 Tubulin, alpha, ubiquitous K-ALPHA-1 174 10416 NM_001428 Enolase 1, (alpha) ENO1 357 9816 NM_006098 Guanine nucleotide binding protein (G protein), beta GNB2L1 45 8910 polypeptide 2-like 1 NM_002032 Ferritin, heavy polypeptide 1 FTH1 138 8861 NM_002654 Pyruvate kinase, muscle PKM2 643 7413 NM_004048 Beta-2-microglobulin B2M 568 7142 NM_006597 Heat shock 70 kDa protein 8 HSPA8 258 6066 NM_000034 Aldolase A, fructose-bisphosphate ALDOA 252 5703 NM_021009 Ubiquitin C UBC 67 5579 NM_006013 Ribosomal protein L10 RPL10 1,503 5572 NM_012423 Ribosomal protein L13a RPL13A 509 5552 NM_007355 Heat shock 90 kDa protein 1, beta HSPCB 309 5436 NM_004046 ATP synthase, H+ transporting, mitochondrial F1 complex, ATP5A1 164 5434 alpha subunit, isoform 1, cardiac muscle NM_000516 GNAS complex locus GNAS 362 4677 NM_001743 Calmodulin 2 (phosphorylase kinase, delta) CALM2 611 4306 NM_005566 Lactate dehydrogenase A LDHA 566 4186 NM_000973 Ribosomal protein L8 RPL8 92 4042 NM_002948 Ribosomal protein L15 RPL15 1,368 3861 NM_000977 Ribosomal protein L13 RPL13 424 3774 NM_002952 Ribosomal protein S2 RPS2 86 3758 NM_005507 Cofilin 1 (non-muscle) CFL1 508 3616 NM_004039 Annexin A2 ANXA2 294 3560 NM_021019 Myosin, light polypeptide 6, alkali, smooth muscle and non-muscle MYL6 209 3512 NM_002300 Lactate dehydrogenase B LDHB 230 3501 NM_003217 Testis enhanced gene transcript (BAX inhibitor 1) TEGT 1,847 3438 NM_002568 Poly(A) binding protein, cytoplasmic 1 PABPC1 445 3241 NM_001015 Ribosomal protein S11 RPS11 85 3220 NM_003973 Ribosomal protein L14 RPL14 156 3198 NM_000969 Ribosomal protein L5 RPL5 78 3167 NM_007104 Ribosomal protein L10a RPL10A 32 3079 NM_001642 Amyloid beta (A4) precursor-like protein 2 APLP2 1,364 3002 NM_001418 Eukaryotic translation initiation factor 4 gamma, 2 EIF4G2 791 2913 NM_002635 Solute carrier family 25 (mitochondrial carrier; SLC25A3 197 2900 phosphate carrier), member 3 NM_001009 Ribosomal protein S5 RPS5 58 2897 NM_000291 Phosphoglycerate kinase 1 PGK1 1,016 2858 NM_001728 Basigin (OK blood group) BSG 769 2827 NM_001658 ADP-ribosylation factor 1 ARF1 1,194 2772 NM_001003 Ribosomal protein, large, P1 RPLP1 39 2770 NM_018955 Ubiquitin B UBB 144 2732 NM_005998 Chaperonin containing TCP1, subunit 3 (gamma) CCT3 255 2709 NM_001967 Eukaryotic translation initiation factor 4A, Isoform 2 EIF4A2 626 2693 NM_001469 Thyroid autoantigen 70 kDa (Ku antigen) G22P1 259 2682 NM_000918 Procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline P4HB 868 2659 4-hydroxylase), beta polypeptide (protein disulfide isomerase; thyroid hormone binding protein p55) NM_002574 Peroxiredoxin 1 PRDX1 323 2604 NM_001020 Ribosomal protein S16 RPS16 78 2573 NM_007363 Non-POU domain containing, octamer-binding NONO 1,119 2557 NM_001022 Ribosomal protein S19 RPS19 63 2533 NM_001675 Activating transcription factor 4 (tax-responsive enhancer ATF4 85 2479 element B67) NM_005617 Ribosomal protein S14 RPS14 78 2465 NM_001664 Ras homolog gene family, member A RHOA 1,045 2426 NM_005801 Putative translation initiation factor SUI1 836 2425 NM_000981 Ribosomal protein L19 RPL19 80 2381 NM_000979 Ribosomal protein L18 RPL18 49 2362 NM_001026 Ribosomal protein S24 RPS24 77 2355 NM_000975 Ribosomal protein L11 RPL11 53 2314 NM_002117 Major histocompatibility complex, class I, C HLA-C 434 2278 NM_004068 Adaptor-related protein complex 2, mu 1 subunit AP2M1 494 2230 NM_006429 Chaperonin containing TCP1, subunit 7 (eta) CCT7 164 2216 NM_022551 Ribosomal protein S18 RPS18 5,538 2208 NM_001013 Ribosomal protein S9 RPS9 73 2113 NM_005594 Nascent-polypeptide-associated complex alpha polypeptide NACA 133 2075 NM_001028 Ribosomal protein S25 RPS25 74 2066 NM_032378 Eukaryotic translation elongation factor 1 delta (guanine EEF1D 76 2051 nucleotide exchange protein) NM_000999 Ribosomal protein L38 RPL38 50 2007 NM_000994 Ribosomal protein L32 RPL32 64 2003 NM_007008 Reticulon 4 RTN4 973 1969 NM_001909 Cathepsin D (lysosomal aspartyl protease) CTSD 834 1940 NM_006325 RAN, member RAS oncogene family RAN 892 1906 NM_003406 Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation YWHAZ 2,013 1892 protein, zeta polypeptide NM_006888 Calmodulin 1 (phosphorylase kinase, delta) CALM1 3,067 1880 NM_004339 Pituitary tumor-transforming 1 interacting protein PTTG1IP 1,985 1837 NM_005022 Profilin 1 PFN1 289 1787 NM_001961 Eukaryotic translation elongation factor 2 EEF2 504 1754 NM_003091 Small nuclear ribonucleoprotein polypeptides B and B1 SNRPB 295 1735 NM_006826 Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase YWHAQ 1,310 1726 activation protein, theta polypeptide NM_002140 Heterogeneous nuclear ribonucleoprotein K HNRPK 1,227 1725 NM_001064 Transketolase (Wernicke-Korsakoff syndrome) TKT 167 1721 NM_021103 Thymosin, beta 10 TMSB10 317 1714 NM_004309 Rho GDP dissociation inhibitor (GDI) alpha ARHGDIA 1,206 1702 NM_002473 Myosin, heavy polypeptide 9, non-muscle MYH9 1,392 1692 NM_000884 IMP (Inosine monophosphate) dehydrogenase 2 IMPDH2 63 1690 NM_001004 Ribosomal protein, large P2 RPLP2 59 1688 NM_001746 Calnexin CANX 2,302 1677 NM_002819 Polypyrimidine tract binding protein 1 PTBP1 1,561 1663 NM_000988 Ribosomal protein L27 RPL27 59 1660 NM_004404 Neural precursor cell expressed, developmentally down-regulated 5 NEDD5 2,090 1654 NM_005347 Heat shock 70 kDa protein 5 (glucose-regulated protein, 78 kDa) HSPA5 1,757 1651 NM_000175 Glucose phosphate isomerase GPI 296 1635 NM_001207 Basic transcription factor 3 BTF3 300 1632 NM_003186 Transgelin TAGLN 405 1612 NM_003334 Ubiquitin-activating enzyme E1 (A1S9T and BN75 temperature UBE1 199 1590 sensitivity complementing) NM_001018 Ribosomal protein S15 RPS15 32 1574 NM_003404 Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase YWHAB 2,088 1523 activation protein, beta polypeptide NM_003753 Eukaryotic translation initiation factor 3, subunit 7 zeta, 66/67 kDa EIF3S7 152 1509 NM_005762 Tripartite motif-containing 28 TRIM28 193 1507 NM_005381 Nucleolin NCL 284 1501 NM_000995 Ribosomal protein L34 RPL34 450 1495 NM_002823 Prothymosin, alpha (gene sequence 28) PTMA 720 1462 NM_002415 Macrophage migration inhibitory factor (glycosylation-inhibiting MIF 117 1459 factor) NM_002128 High-mobility group box 1 HMGB1 1,527 1457 NM_006908 Ras-related C3 botulinum toxin substrate 1 (rho family, RAC1 1,536 1437 small GTP binding protein Rac1) NM_002070 Guanine nucleotide binding protein (G protein), alpha GNAI2 512 1435 inhibiting activity polypeptide 2 NM_001997 Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV) FAU 68 1428 ubiquitously expressed (fox derived); ribosomal protein S30 NM_014390 Staphylococcal nuclease domain containing 1 SND1 556 1422 NM_014764 DAZ associated protein 2 DAZAP2 1,322 1419 NM_005917 Malate dehydrogenase 1, NAD (soluble) MDH1 208 1396 NM_001494 GDP dissociation inhibitor 2 GDI2 785 1395 NM_014225 Protein phosphatase 2 (formerly 2A), regulatory subunit A PPP2R1A 472 1391 (PR 65), alpha isoform NM_001660 ADP-ribosylation factor 4 ARF4 858 1382 NM_001823 Creatine kinase, brain CKB 206 1381 NM_003379 Villin 2 (ezrin) VIL2 1,272 1380 NM_000182 Hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme A HADHA 647 1379 thiolase/enoyl-Coenzyme A hydratase (trifunctional protein), alpha subunit NM_003746 Dynein, cytoplasmic, light polypeptide 1 DNCL1 281 1375 NM_007103 NADH dehydrogenase (ubiquinone) flavoprotein 1, 51 kDa NDUFV1 103 1352 NM_000992 Ribosomal protein L29 RPL29 164 1349 NM_007209 Ribosomal protein L35 RPL35 35 1345 NM_006623 Phosphoglycerate dehydrogenase PHGDH 231 1340 NM_002796 Proteasome (prosome, macropain) subunit, beta type, 4 PSMB4 108 1340 NM_002808 Proteasome (prosome, macropain) 26S subunit, non-ATPase, 2 PSMD2 231 1326 NM_000454 Superoxide dismutase 1, soluble (amyotrophic lateral sclerosis SOD1 346 1323 1 (adult)) NM_003915 RNA binding motif protein 12 RBM12 216 1323 NM_004924 Actinin, alpha 4 ACTN4 1,099 1316 NM_006086 Tubulin, beta 3 TUBB3 296 1314 NM_001016 Ribosomal protein S12 RPS12 56 1304 NM_003365 Ubiquinol-cytochrome c reductase core protein I UQCRC1 126 1303 NM_003016 Splicing factor, arginine/serine-rich 2 SFRS2 1,059 1301 NM_007273 Represser of estrogen receptor activity REA 332 1281 NM_014610 Glucosidase, alpha; neutral AB GANAB 1,652 1280 NM_001749 Calpain, small subunit 1 CAPNS1 514 1270 NM_005080 X-box binding protein 1 XBP1 1,003 1269 NM_005216 Dolichyl-diphosphooligosaccharide-protein glycosyltransferase DDOST 616 1268 NM_004640 HLA-B associated transcript 1 BAT1 237 1262 NM_021983 Major histocompatibility complex, class II, DR beta 4 HLA-DRB1 313 1251 NM_013234 Eukaryotic translation initiation factor 3 subunit k eIF3k 84 1251 NM_004515 Interleukin enhancer binding factor 2, 45 kDa ILF2 384 1249 NM_000997 Ribosomal protein L37 RPL37 50 1244 NM_000801 FK506 binding protein 1A, 12 kDa FKBP1A 1,149 1243 NM_000985 Ribosomal protein L17 RPL17 58 1243 NM_001014 Ribosomal protein S10 RPS10 57 1232 NM_001069 Tubulin, beta 2 TUBB2 194 1230 NM_004960 Fusion (involved in t(12; 16) in malignant liposarcoma) FUS 166 1197 NM_005165 Aldolase C, fructose-bisphosphate ALDOC 432 1195 NM_004930 Capping protein (actin filament) muscle Z-line, beta CAPZB 259 1193 NM_000239 Lysozyme (renal amyloidosis) LYZ 1,016 1190 NM_007263 Coatomer protein complex, subunit epsilon COPE 263 1179 NM_001861 Cytochrome c oxidase subunit IV isoform 1 COX4I1 129 1178 NM_003757 Eukaryotic translation initiation factor 3, subunit 2 beta, 36 kDa EIF3S2 408 1169 NM_005745 B-cell receptor-associated protein 31 BCAP31 438 1166 NM_002743 Protein kinase C substrate 80K-H PRKCSH 337 1158 NM_004161 RAB1A, member RAS oncogene family RAB1A 638 1115 NM_002080 Glutamic-oxaloacetic transaminase 2, mitochondrial GOT2 1,039 1114 (aspartate aminotransferase 2) NM_005731 Actin related protein 2/3 complex, subunit 2, 34 kDa ARPC2 448 1113 NM_006445 PRP8 pre-mRNA processing factor 8 homolog (yeast) PRPF8 173 1110 NM_001867 Cytochrome c oxidase subunit VIIc COX7C 168 1106 NM_002375 Microtubule-associated protein 4 MAP4 1,164 1102 NM_003145 Signal sequence receptor, beta (translocon-associated protein beta) SSR2 492 1099 NM_001788 CDC10 cell division cycle 10 homolog (S. cerevisiae) CDC10 1,015 1094 NM_006513 Seryl-tRNA synthetase SARS 323 1085 NM_003754 Eukaryotic translation initiation factor 3, subunit 5 epsilon, 47 kDa EIF3S5 152 1081 NM_005112 WD repeat domain 1 WDR1 845 1080 NM_004893 H2A histone family, member Y H2AFY 635 1072 NM_004494 Hepatoma-derived growth factor (high-mobility group protein 1-like) HDGF 1,339 1069 NM_001436 Fibrillarin FBL 111 1069 NM_003752 Eukaryotic translation initiation factor 3, subunit 8, 110 kDa EIF3S8 201 1060 NM_003321 Tu translation elongation factor, mitochondrial TUFM 207 1038 NM_001119 Adducin 1 (alpha) ADD1 1,569 1037 NM_005273 Guanine nucleotide binding protein (G protein), beta polypeptide 2 GNB2 386 1030 NM_006755 Transaldolase 1 TALDO1 256 1026 NM_023009 MARCKS-like 1 MARCKSL1 774 1014 NM_002799 Proteasome (prosome, macropain) subunit, beta type, 7 PSMB7 162 1012 NM_002539 Omithine decarboxylase 1 ODC1 343 1009 NM_006801 KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum protein retention KDELR1 742 1007 receptor 1 NM_014944 Calsyntenin 1 CLSTN1 1,481 1003 NM_007262 Parkinson disease (autosomal recessive, early onset) 7 PARK7 253 1002 The above list of the accession number for housekeeping genes was obtained from Eisenberg and Levanon (2003). The accession numbers were used as input for a PERL (Programmed Extraction Report Language) computer program that extracts EST data from the Unigene database. The Unigene database was downloaded as a text file from the NCBI website. The length of the 3′UTR was derived by computationally extracting the 3′UTR (Bakheet al, 2001). ^(a)is a commonly used abbreviation of the gene product; ^(b)is the length of the 3′UTR; ^(c)is the number of ESTs.

Example 2

A standard pUC 19 based expression vector containing green fluorescence protein (GFP) was used, which contains the human cytomegalovirus (CMV) immediate early promoter, the coding region of enhanced GFP (EGFP) gene and 3′UTR of bovine growth hormone, and ter contains the polyA signal. Suitable expression vectors could, for example, be purchased from Invitrogen, Clontech, Invivogen, Gene Therapy Systems, and Promega, Inc. A PCR product derived from a portion of the 3′UTR of the human elongation factor alpha 1, EEF1A, was generated using a forward primer that contains a BamHI restriction site at the 5′ terminus of the primer (SEQ ID NO: 1) and a reverse primer that contains a XbaI restriction site at the 5′ terminus of the primer (SEQ ID NO: 2). The sequences of the primers are as follows:

     BamHI GCACC GGATCC AATATTATCCCTAATACCTG      XbaI GCCAG TCTAGA AATAACTTAAAACTGCCA

The primers amplify a 210 bp region 1461-1680 in EEF1A which has the accession number NM_(—)001402. The PCR product was cut using restriction enzymes BamHI and XbaI (New England BioLabs, NEB). Briefly, 10 μg of the PCR product was digested with 10 units of XbaI in a buffer containing 0.1 μg/ml BSA for 1 hr at 37° C. followed by digestion with BamHI in BamHI buffer for an additional 1 hr at 37° C. The digested PCR products were extracted using a phenol/chloroform method followed by ethanol precipitation. The PCR region was cloned into the GFP vector that was previously digested with the same restriction enzymes (BamHI and XbaI) and previously purified using phenol/chloroform extraction and ethanol precipitation. The BamHI and XbaI sites are located downstream of the end of the EGFP coding region and upstream of the polyA signal (FIG. 1A), Cloning of the PCR products into the vector was achieved using a ligation reaction: 30 ng of digested vector DNA is mixed with 90 ng of digested PCR products in a 10 μl reaction containing T4 DNA ligase. The ligated products were used to transform competent DH5α E. coli cells followed by expansion of the resulting colonies in bacterial culture medium. The recombinant DNA was extracted using the Qiagen plasmid purification kit (Qiagen, Germany). The sizes of the vectors harboring the inserts were verified using gel electrophoresis. The size of the vector without the modified 3′UTR is 5757 bases and the size of the vector with the modified 3′UTR is 5933 bases.

The recombinant hybrid 3′UTR sequence (˜400 bp) was searched against NCBI human genome databases to search for the best homology and found to contain the following: a portion of the human EEFA1 and a portion of bovine growth hormone bGH) containing polyA signal. The sequence of the entire hybrid 3′UTR is given in Table 2 (SEQ ID NO: 3).

TABLE 2 Sequence of the efficient hybrid 3′UTR ggatccaaatattatccctaatacctgccaccccactcttaatcagtggtggaagaacggtc tcagaactgtttgtttcaattggccatttaagtttagtagtaaaagactggttaatgataac aatgcatcgtaaaaccttcagaaggaaaggagaatgttttgtggaccactttggttttcttt tttgcgtgtggcagttttaagttattctctagagatctgtgtgttggttttttgtggatctg ctgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctg gaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgag taggtgtcattctattctggggggtggggtggggcagcacagcaagggggaggattgggaag acaatagcaggcatgctggggatgcggtgggctctatgggta The polyA site (AATAAA) is underlined.

The resultant expression vector with the hybrid 3′UTRs were used for functional studies to confirm the expression of the encoded protein, namely GFP. The HEK 293 cell line was used for transfection. HEK293 cells were grown at standard culture conditions (37° C., 5% CO₂) in RPMI 1788 medium supplemented with 10% PBS and antibiotics (GIBCO BRL, Gaithersburg, Md.). 3×10⁴ cells per well in 96-well plates were transfected with 1 μg of the original (unmodified) expression vector or the modified vector. Transfections were performed in serum-free medium using LipofectinAmine 2000 (Gibco) for 5 h, followed by replacing medium with serum-supplemented medium. After approximately 48 hours, the cells were visualized using a fluorescence microscope and the optimal excitation wavelength for GFP of 488 nm and emission wavelength of 503 nm. Images were captured using a camera mounted on top of the microscope. The images were read by an algorithm that quantitates total fluorescence intensities in pixels. FIG. 2 shows exemplary images and the fluorescence results of these images. The modified vector, i.e. the vector with the hybrid 3′UTR, resulted in an approximately 3-fold increase in the protein expression as evaluated by the GFP expression.

Example 3

The expression vector with the hybrid 3′UTR was used in two types of cell lines, HeLa cells which is a cervical cell line (FIGS. 3A and B), and Huh7 which is a liver cell line (FIGS. 3C and D). The cells were grown at standard culture conditions (37° C., 5% CO₂) in DMEM medium supplemented with 10% FBS and antibiotics (Gibco BRL, Gaithersburg, Md.). 3×10⁴ cells per well in 96-well plates were transfected with 1 μg of the original expression vector (FIGS. 3A and C) or the modified vector FIGS. 3B and D). Transfections were performed in serum-free medium using LipofectinAmine 2000 (Gibco) for 5 h, followed by replacing medium with serum-supplemented medium. After approximately 48 hours, the cells were visualized using a fluorescence microscope and the optimal excitation wavelength for GFP of 488 nm and emission wavelength of 503 nm. Images were captured using a camera mounted on top of the microscope. The images were read by an algorithm that quantitated total fluorescence intensities in pixels. FIG. 3 shows the results of the quantitation. The modified vector, i.e. the vector with the hybrid 3′UTR, resulted in an approximately 5-fold increase in the protein expression in HeLa cell line and more than 15-fold increase in protein expression in Huh7 cell line (as evaluated by the GFP expression).

Example 4

A PCR product generated from the modified vector using primers flanking the CMV promoter and the hybrid 3′UTR still leads to efficient transfection and optimum protein expression. HEK293 cells in 3×10⁴ cells per well in 96-well plates were transfected with 1 μg of the original expression vector (FIG. 4A) or with said PCR product generated from the modified vector (FIG. 4B). Transfections were performed in serum-free medium using LipofectinAmine 2000 (Gibco) for 5 h, followed by replacing medium with serum-supplemented medium. After approximately 72 hours, the cells were visualized using a fluorescence microscope and the optimal excitation wavelength for GFP of 488 nm and emission wavelength of 503 nm. Images were captured using a camera mounted on top of the microscope. The images were read by an algorithm that quantitates total fluorescence intensities in pixels. FIG. 4 also shows the results of the quantitations showing that the GFP expression resulting from the PCR product which contains the hybrid 3′UTR is still as effective as the GFP expression resulting from the original vector.

REFERENCES

-   Bakheet, T., Frevel, M., Williams, B R, and K. S. Khabar, 2001.     APED: Human AU-rich element-containing mRNA database reveals     unexpectedly diverse functional repertoire of encoded proteins.     Nucleic Acids Research. 29:246-254. -   Chen, C. Y. and A. D. Shyu. 1994. Selective degradation of     early-response-gene mRNAs: functional analyses of sequence features     of the AU-rich elements. Mol Cell Biol 14: 8471-8482. -   Dalphin, M. E., P. A. Stockwell, W. P, Tate, and C. M. Brown. 1999,     TransTerm, the translational signal database, extended to include     full coding sequences and untranslated regions. Nucleic Acids Res     27: 293-294. -   Eisenberg, E. and E. Y. Levanon. 2003. Human housekeeping genes are     compact. Trends Genet. 19(7): 362-365. -   Foecking, M. K. and H. Hofstetter. 1986. Powerful and versatile     enhancer-promoter unit for mammalian expression vectors. Gene 45:     101-105. -   Goodwin B. C. and F. M. Rottman. 1992. The 3′-flanking sequence of     the bovine growth hormone gene contains novel elements required for     efficient and accurate polyadenylation. J Biol Chem 267:     16330-16334. -   Kobayashi, M., A. Tanaka, Y. Hayashi, and S. Shimamura. 1997. The     CMV enhancer stimulates expression of foreign genes from the human     BF-1 alpha promoter. Anal Biochem 247: 179-181. -   Legendre, M. and D. Gautheret. 2003. Sequence determinants in human     polyadenylation site selection. BMC Genomics 4: 7. -   Shaw, G. and R. Kramen. 1986. A conserved AU sequence from the 31     untranslated region of GM-CSF mRNA mediates selective mRNA     degradation. Cell 46: 659-667. -   Wigley, P. L., M. D. Sheets, D. A. Zarkower, M. E. Whitner, and M.     Wickens. 1990. Polyadenylation of mRNA: minimal substrates and a     requirement for the 2′ hydroxyl of the U in AAUAAA. Mol Cell Biol     10: 1705-1713. 

The invention claimed is:
 1. A recombinant nucleic acid comprising a hybrid 3′ untranslated region (3′UTR) that comprises a first and a second nucleic acid region, wherein (a) said first region is a 3′ untranslated region (3′UTR) of a stable eukaryotic mRNA of eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) or is part of the 3′UTR of EEF1A1 mRNA, (b) said first region does not contain a polyadenylation signal, (c) said second region is derived from the downstream end of an 3′ untranslated region of another or the same stable eukaryotic mRNA as in (a), and (d) said second region contains a polyadenylation signal, wherein said recombinant nucleic acid has the sequence of SEQ ID NO:3.
 2. The recombinant nucleic acid of claim 1, wherein said first region and said second region are in proximity to each other or are adjacent to each other or overlap with each other or one encompasses the other.
 3. The recombinant nucleic acid of claim 2, wherein the 3′ end of the first region is adjacent to the 5′ end of the second region or the 3′ end of the second region is adjacent to the 5′ end of the first region or the first region is located within the second region.
 4. The recombinant nucleic acid of claim 1, wherein the nucleic acid is DNA, RNA, or PNA.
 5. An expression vector comprising the recombinant nucleic acid according to claim
 1. 6. The expression vector according to claim 5 further comprising a selection marker.
 7. The expression vector according to claim 5, wherein the recombinant nucleic acid according to claim 1 further comprises a protein coding sequence that encodes a reporter protein or a therapeutic protein.
 8. An isolated host cell comprising the expression vector of claim 5 wherein the expression vector of claim 5 comprises a protein coding sequence, and the host cell transiently or encoded in the expression vector of claim
 5. 9. An isolated host cell which is obtained by in vivo injection of the expression vector of claim 5 into a cell, wherein the expression vector of claim 5 comprises a protein coding sequence, and the host cell comprises the expression vector of claim 5 and transiently or encoded in the expression vector of claim
 5. 10. A method for obtaining a host cell, comprising the following steps of (a) providing an expression vector according to claim 5, (b) in vivo injection of the expression vector according to claim 5 into a cell, and (c) obtaining the injected cell of step (b).
 11. A method for expressing proteins, comprising providing and culturing of the host cell according to claim 8, under conditions allowing transient or stable expression of proteins, and obtaining said expressed proteins.
 12. The recombinant nucleic acid of claim 3, wherein the first region is located upstream of the polyadenylation signal of the second region.
 13. A recombinant nucleic acid comprising the following components in 5′ to 3′ direction: (a) a promoter sequence, (b) a protein coding region sequence, and (c) a hybrid 3′UTR comprising the sequence of SEQ ID NO: 3, which is operably linked to the protein coding sequence of (b).
 14. The recombinant nucleic acid according to claim 13, wherein the nucleic acid is a linear DNA molecule and is generated by PCR using at least two primers that specifically hybridize to regions near the 5′ end and specifically hybridize to regions near the 3′ end of the nucleic acid according to claim 13 which is comprised in an expression vector.
 15. A method of producing a nucleic acid in linear form, comprising the following steps of (a) providing an expression vector comprising the recombinant nucleic acid according to claim 13, (b) providing at least two primers, wherein one primer specifically hybridizes to regions near the 5′ end of the nucleic acid according to claim 6, and wherein the other primer specifically hybridizes to regions near the 3′ end of the nucleic acid according to claim 13, (c) performing a PCR, and (d) obtaining the amplification product of step (c).
 16. An isolated host cell comprising the recombinant nucleic acid of claim 14, wherein the host cell is capable of transiently or stably expressing the protein encoded by the recombinant nucleic acid of claim
 14. 17. An isolated host cell which is obtained by in vivo injection of the recombinant nucleic acid of claim 14 into a cell, wherein the host cell comprises the recombinant nucleic acid of claim 14 and is capable transiently or stably expressing the protein encoded by the recombinant nucleic acid of claim
 14. 18. A method for obtaining a host cell, comprising the following steps of (a) providing a recombinant nucleic acid of claim 14, (b) in vivo injection of the recombinant nucleic acid of claim 14 into a cell, and (c) obtaining the injected cell of step (b).
 19. A method for expressing proteins, comprising providing and culturing of the host cell according to claim 16, under conditions allowing transient or stable expression of proteins, and obtaining said expressed proteins. 